The kinesin molecular motors are required for a wide range of cellular functions including vesicle trafficking, spindle assembly and chromosome segregation, as well as signal transduction. These ATPases can pull a cellular cargo along a microtubule, slide one microtubule relative to another, or even remodel the microtubule cytoskeleton by promoting microtubule disassembly. The goal of this proposal is to establish the kinetic and thermodynamic basis of force generation for the Eg5 and Kar3Cik1 kinesin ATPases in comparison to conventional kinesin and Ned. Eg5, Ned, and Kar3 are involved in spindle dynamics during meiosis and/or mitosis, and Kar3 is essential for karyogamy (nuclear fusion) during conjugation. Functional roles in vivo require movement of microtubules relative to each other, and Kar3 also exhibits a microtubule depolymerizing activity. Ned and Kar3 promote microtubule minus-end directed force generation, yet Eg5 and kinesin drive plus-end directed motion. Eg5 functions as a homotetramer, yet Kar3 associates with Cik1, a non-motor protein, to form a heterodimer. Kinesin is a processive motor, yet Eg5, Ned, and Kar3 are believed not to be processive. The specific aims are directed to determine the role of Cik1 for Kar3Cik1 mechanochemistry, to examine the mechanochemistry and structural transitions of Kar3Cik1 at conditions where the motor slides one microtubule relative to another in direct comparison to conditions where Kar3Cik1 promotes microtubule disassembly, and to determine the interactions of Cik1 with the microtubule independent of Kar3. For Eg5, the proposed research will define the cooperative interactions between the motor domains of a dimer and will address how the structural changes are coordinated during the ATPase cycle. A comprehensive analysis of these 4 kinesins will provide new information to begin to understand the structural and mechanistic requirements for the diverse movements occurring during the cell cycle where genetic alteration can result in birth defects and diseases such as cancer.